Cytokine production inhibitors, agents for protecting and promoting liver function, anti-inflammatory agents, immunosuppressants, drugs, cosmetics, foods and food materials

ABSTRACT

Highly safe cytokine production inhibitors, agents for protecting and promoting liver function, anti-inflammatory agents and immunosuppressants. Brazilian licorice extract and periandrins, which are constituents thereof, have excellent characteristics of showing effects of inhibiting cytokine production and protecting and promoting liver function, an anti-inflammatory effect and an immunosuppressive effect without any harmful side effects. Thus use of Brazilian licorice extract or periandrins as cytokine production inhibitors makes it possible to inhibit inflammations in various diseases such as rheumatoid arthritis. These substances are also usable as agents for protecting and promoting liver function, anti-inflammatory agents and immunosuppressants. Moreover, foods, cosmetics, sweeteners and food materials containing Brazilian licorice extract exert the effects as described above.

TECHNICAL FIELD OF THE INVENTION

This invention relates to cytokine production inhibitors, agents forprotecting and promoting liver function, anti-inflammatory agents,immunosuppressants, drugs, cosmetics, foods, and food materials.

BACKGROUND OF THE INVENTION

Inflammation by autoimmune diseases like rheumatism, allergies andatopies is promoted by production of migration enhancement factors ofleukocytes called cytokine.

In order to inhibit such cytokine production, steroids such asprednisolone have been conventionally used.

However, steroids have strong side effects. Therefore, repeatedadministration of the same is difficult.

To make matters worse, there is no report on any component of edibleplants and galenicals which shows strong effect of inhibiting cytokineproduction even at the basic research level.

In short, hardly any medical agent has been existed which can inhibitcytokine production without any side effects, and such an agent has beenexpected to be discovered.

Licorice (leguminous perennial herb) and glycyrrhizin which is acomponent thereof have been used for an anti-inflammatory agent,anti-allergic agent and agent for protecting liver function, whichpromote detoxication and tissue recovery.

However, since glycyrrhizin inhibits corticosteroid metabolism,pseudoaldosteronism (edema with sodium retention, hypertension) may bedeveloped as a side effect.

Therefore, a licorice cannot be administered over a long period to apatient having kidney diseases or hypertension.

Accordingly, a medical agent which is as effective as licorice but whichdoes not develop any side effect has been expected.

SUMMARY OF THE INVENTION

One object of the present invention is to provide a medical agent whichexerts effects of inhibiting cytokine production, protecting andpromoting liver function, an anti-inflammatory effect, and animmunosuppressive effect, and which hardly causes any side effect.Another object of the present invention is to provide drugs, cosmetics,foods, and food materials to which the aforementioned medical agent isapplied.

Heretofore, periandrins and periandradulcins have been reported as maincomponents of a Brazilian licorice. These are both glycosidesstructurally similar to glycyrrhizin which is a component of a licorice,and are expected to act similar to glycyrrhizin. However, hardly anyreport has been made in relation to their physiological activities.

The inventors discovered that Brazilian licorice extract andperiandrins, which are constituents thereof, have an anti-inflammatoryeffect and an immunosuppressive effect, and accomplished the presentinvention.

Brazilian licorice extract is, for example, obtained using extractionvehicles such as ethyl alcohol.

The invention provides a cytokine production inhibitor comprisingperiandrins as an active ingredient.

Since the periandrins show an effect of inhibiting cytokine production,the cytokine production inhibitor of the present invention inhibitscytokine production and acts as an anti-inflammatory agent.

Accordingly, the cytokine production inhibitor of the present inventioncan inhibit inflammation developed by cell disorders (for example, bybacteria, toxic elements in medicine and alcohol), autoimmune diseases(such as rheumatism (rheumatoid arthritis, for example), systemic lupuserythematosus, collagenosis, etc.), atopies, allergies, and pollinosis.

Furthermore, periandrins do not inhibit activation of 11β-hydroxysteroid dehydrogenase.

If activation of 11 β-hydroxysteroid dehydrogenase is inhibited,corticosteroid metabolism is also inhibited, resulting in thatpseudoaldosteronism (edema with sodium retention, hypertension) mayoccur.

Accordingly, the cytokine production inhibitor of the present inventioncomprising periandrins does not cause pseudoaldosteronism and can beadministered, for example, even to a patient having kidney diseases andhypertension over a long period.

The cytokine production inhibitor of the present invention can beproduced by compounding periandrins obtained by separating a componentheld by adsorption chromatography carrier from the Brazilian licoriceextract, for example.

In this case, since polysaccharide contained in the Brazilian licoriceextract is not entered into the cytokine production inhibitor, it isdifficult for the cytokine production inhibitor to get moldy.

Examples of the aforementioned periandrins are periandrin I (PD-I),periandrin II (PD-II), periandrin III (PD-III), periandrin IV (PD-IV),etc.

Cytokine is a cellular glycoprotein, and an endogenous substance in vivowhich acts on the other cells. It is a general term for interleukin,interferon, lymphokine, monokine, and tumor necrosis factors.

When cells are damaged by bacteria, toxic elements in medicine, alcohol,etc., or in case of autoimmune diseases such as rheumatism,collagenosis, etc., phagocyte such as macrophage, etc. is activated andautopepsia occurs, resulting in that inflammation is developed. At thesame time, cytokine which is a type of migration enhancement factors ofleukocytes is produced. However, cytokine has a number of factors whichaccelerates immunoreaction. Therefore, if cytokine is produced in alarge amount, it accelerates aggregation of phagocyte and furtherworsens the inflammation.

The invention provides a cytokine production inhibitor containing one ofBrazilian licorice extract and one or more constituents contained in theextract as an active ingredient.

The cytokine production inhibitor of the present invention contains anextract, or one or more constituents contained in the same, obtained byfiltering and drying the liquid extracted from a dried rootstalk of aBrazilian licorice with alcohol.

Brazilian licorice extract inhibits cytokine production, and thus thecytokine production inhibitor of the present invention inhibits cytokineproduction and serves as an anti-inflammatory agent.

Inflammation which is a symptom of diseases such as cell disorders (forexample, by bacteria, toxic elements in medicine and alcohol),autoimmune diseases (such as rheumatism (rheumatoid arthritis, forexample), systemic lupus erythematosus, collagenosis, etc.), atopies,allergies, and pollinosis is in many cases accelerated by cytokine.Consequently, the cytokine production inhibitor of the present inventioninhibits inflammation by the aforementioned diseases.

Brazilian licorice extract, in addition, is on the list of foodadditives in Japan as a sweetener, and is superior in that it developsno harmful side effect.

The invention provides the cytokine production inhibitor one or moreconstituents of the aforementioned comprise periandrins.

The cytokine production inhibitor of the present invention inhibitscytokine production due to periandrins contained in Brazilian licoriceextract.

Accordingly, the cytokine production inhibitor of the present invention,inhibits inflammation developed by cell disorders, autoimmune diseases,atopies, allergies, and pollinosis, which may be worsened by cytokine.

This cytokine production inhibitor does not inhibit activation of 11β-hydroxysteroid dehydrogenase, and thus does not causepseudoaldosteronism and can be administered, for example, to a patienthaving kidney diseases and hypertension, over a long period.

Furthermore, this cytokine production inhibitor has no harmful sideeffect. Therefore, it can be administered over a long period intreatment of autoimmune diseases and after organ transplant, forexample, without developing any side effect unlike the conventionalsteroids.

The invention provides drugs comprising the cytokine productioninhibitor as an anti-inflammatory, anti-allergic, or anti-atopic activeingredient.

The drugs of the present invention comprise periandrins, Brazilianlicorice extract, or one or more constituents of the extract. Therefore,it inhibits cytokine production and eases symptoms of inflammation,allergy, atopy, and pollinosis.

The drugs of the present invention are also advantageous in that theyhave no side effect.

The drugs of the present invention can be taken as oral medicine,external preparations, injectable solutions, vaparole, nose drops, eyedrops, etc., for example.

The drugs of the present invention can be in the form of pill or tablet,liquid, injectable solution, ointment, cream, lotion, aerosol,suppository, etc., for example.

The drugs of the present invention can compounded with other ingredientswhich show an anti-inflammatory effect, an anti-allergic effect, or ananti-atopic effect.

The invention provides cosmetics comprising the cytokine productioninhibitor as an anti-inflammatory, anti-allergic, or anti-atopic activeingredient.

The cosmetics of the present invention comprise periandrins, Brazilianlicorice extract, or one or more constituents of the extract. Therefore,it inhibits cytokine production and eases symptoms of inflammation,allergy, atopy, and pollinosis.

Accordingly, use of the cosmetics of the present invention can improveitches and pains on the skin by allergic symptoms and inflammation. Thecosmetics of the present invention can be used even by an atopic person.

The cosmetics of the present invention are also advantageous in thatthey have no side effect.

The cosmetics of the present invention can be in the form of cream,ointment, lotion, milky lotion, solid body, powder, etc., for example.

Examples of the cosmetics of the present invention are basic skin careproducts such as lotion, milky lotion, essence, moisturizing cream,etc., sun protection products such as sunscreen cream, sunscreen lotion,sunscreen oil, carmine lotion, etc., makeups such as foundation, eyeliner, mascara, eye shadow, blush-on for cheeks, lipstick, etc.,cleansers such as facial wash, body shampoo, hair shampoo, etc,perfumes, antiperspirant deodorants, etc.

The invention provides foods comprising the cytokine productioninhibitor as an anti-inflammatory, anti-allergic, or anti-atopic activeingredient.

The foods of the present invention contain periandrins, Brazilianlicorice extract, or one or more constituents of the extract. Therefore,it inhibits cytokine production and eases symptoms of inflammation,allergy, atopy, and pollinosis.

Accordingly, an intake of the foods of the present invention can improvevarious inflammatory symptoms by atopy, pollinosis, etc., for example.

The foods of the present invention are also advantageous in that theyhave no side effect.

The foods of the present invention can be made into oral compositionssuch as tea, soft drinks, gums, candies, etc., pastes of marine productssuch as steamed fish pastes, fish sausages, etc., stock farm productssuch as sausages, hams, etc., Western-style confectionery,Japanese-style confectionery, noodles such as raw noodles, noodles forboiling, etc., seasonings such as sauces, soy sauces, dips, etc., andgeneral food products such as pickles, prepared foods, etc.

The foods of the present invention can, as far as the effect of thepresent invention is not deteriorated, compound various ingredientsgenerally used in foods, such as sugar, condensed milk, wheat flour,shortening, salt, glucose sugar, chicken eggs, butter, margarine, starchsyrup, calcium, iron, vitamins, seasonings, spices, etc.

The invention provides food materials comprising the cytokine productioninhibitor as an anti-inflammatory, anti-allergic, or anti-atopic activeingredient.

The food materials of the present invention comprise periandrins,Brazilian licorice extract, or one or more constituents of the extract.Therefore, it inhibits cytokine production and eases symptoms ofinflammation, allergy, atopy, and pollinosis.

Accordingly, an intake of foods compounding the food materials of thepresent invention can improve various inflammatory symptoms by atopy,pollinosis, etc., for example.

The food materials of the present invention are also advantageous inthat they have no side effect.

The food materials of the present invention can be a sweetener, forexample.

The food materials of the present invention can compound variousingredients generally used in foods (like sugar, condensed milk, wheatflour, shortening, salt, glucose sugar, chicken eggs, butter, margarine,starch syrup, calcium, iron, vitamins, seasonings, spices, etc.).

The food materials of the present invention can be added to variousfoods and drinks (like oral compositions such as tea, soft drinks, gums,candies, etch, pastes of marine products such as steamed fish pastes,fish sausages, etc., stock farm products such as sausages, hams, etc.,Western-style confectionery, Japanese-style confectionery, noodles suchas raw noodles, noodles for boiling, etc., seasonings such as sauces,soy sauces, dips, etc., and general food products such as pickles,prepared foods, etc.).

The invention provides an agent for protecting and promoting liverfunction comprising periandrins as an active ingredient.

The periandrins show an effect of protecting and promoting liverfunction, and thus the agent for protecting and promoting liverfunction, comprising periandrins as an active ingredient, of the presentinvention has also an effect of protecting and promoting liver function.

Cytokine is produced in case that there are any liver disorders.However, the agent for protecting and promoting liver function of thepresent invention inhibits cytokine production by protecting andpromoting liver function and eases symptoms of inflammation, allergy,atopy, and pollinosis, for example.

Accordingly, the agent for protecting and promoting liver function ofthe present invention is as effective as the conventionally usedlicorice extract, and more advantageously, it has no side effect unlikelicorice.

Therefore, the agent for protecting and promoting liver function of thepresent invention can be administered over a long period, for example,to a patient who has kidney diseases and hypertension and to whomlong-term administration of licorice is not possible.

The agent for protecting and promoting liver function of the presentinvention can compound periandrins separated and purified from Brazilianlicorice extract, for example. In this case, since polysaccharidecontained in Brazilian licorice extract is not entered into the agentfor protecting and promoting liver function, it is difficult for theagent for protecting and promoting liver function to get moldy.

Examples of the aforementioned periandrins are periandrin I (PD-I),periandrin II (PD-II), periandrin III (PD-III), periandrin IV (PD-IV),etc.

The invention provides an agent for protecting and promoting liverfunction comprising one of Brazilian licorice extract and one or moreconstituents contained in the extract as an active ingredient.

The agent for protecting and promoting liver function of the presentinvention comprises extract, or one or more constituents contained inthe same, obtained by filtering and drying liquid extracted from a driedrootstalk of a Brazilian licorice with alcohol.

Brazilian licorice extract shows an effect of protecting and promotingliver function, and thus the agent for protecting and promoting liverfunction, comprising Brazilian licorice extract as an active ingredient,of the present invention is also effective in protecting and promotingof liver function.

Cytokine is produced in case that there are any liver disorders.However, the agent for protecting and promoting liver function of thepresent invention inhibits cytokine production by protecting andpromoting liver function and eases symptoms of inflammation, allergy,atopy, and pollinosis, for example.

Accordingly, the agent for protecting and promoting liver function ofthe present invention is as effective as the conventionally usedlicorice extract. More advantageously, it has no side effect unlikelicorice.

Therefore, the agent for protecting and promoting liver function of thepresent invention can be administered over a long period, for example,to a patient who has kidney diseases and hypertension and to whomlong-term administration of licorice is not possible.

The invention provides the agent for protecting and promoting liverfunction one or more constituents of the aforementioned compriseperiandrins.

The agent for protecting and promoting liver function of the presentinvention comprises periandrins contained in the extract as an activeingredient.

Therefore, this agent for protecting and promoting liver function,inhibits cytokine production by protecting and promoting liver functionand eases symptoms of inflammation, allergy, atopy, and pollinosis, forexample.

Also this agent for protecting and promoting liver function has no sideeffect (such as pseudoaldosteronism).

Therefore, it can be administered over a long period, for example, to apatient who has kidney diseases and hypertension and to whom long-termadministration of licorice is not possible.

The invention provides drugs comprising the agent for protecting andpromoting liver function as an active ingredient for protecting andpromoting liver function.

The drugs of the present invention comprise periandrins, Brazilianlicorice extract, or one or more constituents contained in the extract.Thus, they have an effect of protecting and promoting liver function.

Also, the drugs of the present invention inhibit cytokine production byprotecting and promoting liver function and ease symptoms ofinflammation, allergy, atopy, and pollinosis, for example.

The drugs of the present invention are also advantageous in that theyhave no harmful side effect.

The drugs of the present invention, as well as the drugs can be madeinto various forms and compounded with other ingredients.

The invention provides foods comprising the agent for protecting andpromoting liver function as an active ingredient for protecting andpromoting liver function.

The foods of the present invention comprise periandrins, Brazilianlicorice extract, or one or more constituents contained in the extract.Thus, they have effect of protecting and promoting liver function.

Also, the foods of the present invention inhibit cytokine production byprotecting and promoting liver function and ease symptoms ofinflammation, allergy, atopy, and pollinosis, for example.

Accordingly, an intake of the foods of the present invention can protectand promote liver function and improve various inflammatory symptoms byatopy, pollinosis, etc., for example.

The foods of the present invention are also advantageous in that theyhave no harmful side effect.

The foods of the present invention, can be made into various forms andcompounded with other ingredients.

The invention provides food materials comprising the agent forprotecting and promoting liver function as an active ingredient forprotecting and promoting liver function.

The food materials of the present invention comprise periandrins,Brazilian licorice extract, or one or more constituents contained in theextract. Thus, they have an effect of protecting and promoting liverfunction.

Also, the food materials of the present invention inhibit cytokineproduction by protecting and promoting liver function and ease symptomsof inflammation, allergy, atopy, and pollinosis, for example.

Accordingly, an intake of foods compounding the food materials of thepresent invention can protect and promote liver function and improvevarious inflammatory symptoms by atopy, pollinosis, etc., for example.

The foods of the present invention are also advantageous in that theyhave no harmful side effect.

The food materials of the present invention can be a sweetener, forexample.

The food materials of the present invention, can be compounded withvarious ingredients and can be added to various foods.

The invention provides an anti-inflammatory agent comprising periandrinsas an active ingredient.

The anti-inflammatory agent of the present invention compriseperiandrins, and thus, it shows an anti-inflammatory effect.

Particularly, the anti-inflammatory agent of the present invention canreduce inflammation due to autoimmune diseases (such as rheumatism(rheumatoid arthritis, for example), systemic lupus erythematosus,couagenosis, multiple sclerosis, and atopies) by inhibiting cytokineproduction.

The anti-inflammatory agent of the present invention is alsoadvantageous in that they have no harmful side effect. Therefore, it canbe administered over a long period in treatment of autoimmune diseasesand after organ transplant, for example, without developing any sideeffect unlike the conventional steroids.

The anti-inflammatory agent of the present invention can compoundperiandrins separated and purified from Brazilian licorice extract, forexample. In this case, since polysaccharide contained in Brazilianlicorice extract is not entered into the agent for protecting andpromoting liver function, it is difficult for the agent for protectingand promoting liver function to get moldy.

Examples of the aforementioned periandrins are periandrin I (PD-I),periandrins II (PD-II), periandrin III (PD-III), periandrin IV (PD-IV),etc.

The invention provides an anti-inflammatory agent comprising one ofBrazilian licorice extract and one or more constituents contained in theextract as an active ingredient.

The anti-inflammatory agent of the present invention comprises theextract, or one or more constituents contained in the same, and thus, itshows an anti-inflammatory effect.

Particularly, the anti-inflammatory agent of the present invention canreduce inflammation due to autoimmune diseases (such as rheumatism(rheumatoid arthritis, for example), systemic lupus erythematosus,collagenosis, multiple sclerosis, and atopies) by inhibiting cytokineproduction.

The anti-inflammatory agent of the present invention is alsoadvantageous in that they have no harmful side effect. Therefore, it canbe administered over a long period in treatment of autoimmune diseasesand after organ transplant, for example, without developing any sideeffect unlike the conventional steroids.

Examples of the aforementioned one or more constituents are triterpenoidglycosides and triterpenoid aglycones, such as periandrins I, II, III,IV and V, periandradulcins A, B and C, and aglycones of theaforementioned. In short, they are triterpenoid glycosides and/ortriterpenoid aglycones contained in Brazilian licorice extract.

The invention provides the anti-inflammatory agent one or moreconstituents of the aforementioned comprise periandrins.

The anti-inflammatory agent of the present invention comprisesperiandrins contained in Brazilian licorice extract, and thus, it showsan anti-inflammatory effect.

Also, the anti-inflammatory agent of the present invention, can reduceinflammation due to autoimmune diseases (such as rheumatism (rheumatoidarthritis, for example), systemic lupus erythematosus, collagenosis,multiple sclerosis, and atopies) by inhibiting cytokine production.

The anti-inflammatory agent of the present invention is alsoadvantageous in that they have no harmful side effect. Therefore, it canbe administered over a long period in treatment of autoimmune diseasesand after organ transplant, for example, without developing any sideeffect unlike the conventional steroids.

The invention provides an immunosuppressant comprising periandrins as anactive ingredient.

Also, the immunosuppressant, comprising periandrins, of the presentinvention can ease symptoms of autoimmune diseases (caused byabnormality in human immune system mistaking cells or tissues of itselffor antigens due to genetic predisposition and low molecular extrinsicfactor called hapten (such as, rheumatism (rheumatoid arthritis, forexample), systemic lupus erythematosus, collagenosis, multiplesclerosis, and atopies).

Specifically, this autoimmune agent is unlikely to cause aggravation(i.e. rebound) of the symptoms after the administration is stopped.

Additionally, since the autoimmune agent of the present invention, hasno harmful side effect, it can be administered over a long period.

The autoimmune agent of the present invention can compound periandrinsseparated and purified from Brazilian licorice extract, for example. Inthis case, since polysaccharide contained in Brazilian licorice extractis not entered into the agent for protecting and promoting liverfunction, it is difficult for the agent for protecting and promotingliver function to get moldy.

Examples of the aforementioned periandrins are periandrin I (PD-I),periandrins II (PD-II), periandrin III (PD-III), periandrin IV (PD-IV),etc.

The invention provides an immunosuppressant comprising one of Brazilianlicorice extract and one or more constituents contained in the extractas an active ingredient.

The immunosuppressant, comprising one of Brazilian licorice extract andone or more constituents contained in the extract, of the presentinvention can ease symptoms of autoimmune diseases (caused byabnormality in human immune system mistaking cells or tissues of itselffor antigens due to genetic predisposition and low molecular extrinsicfactor called hapten (such as, rheumatism (rheumatoid arthritis, forexample), systemic lupus erythematosus, collagenosis, multiplesclerosis, and atopies).

Also, the autoimmune agent of the present invention has no harmful sideeffect, and thus it can be administered over a long period.

Examples of the aforementioned one or more constituents are triterpenoidglycosides and triterpenoid aglycones, such as periandrins I, II, III,IV and V, periandradulcins A, B and C, and aglycones of theaforementioned. In short, they are triterpenoid glycosides and/ortriterpenoid aglycones comprised in Brazilian licorice extract.

The invention provides the autoimmune agent and one or more constituentsof the aforementioned comprise periandrins.

The immunosuppressant, comprising periandrins as one or moreconstituents contained in Brazilian licorice extract, of the presentinvention can ease symptoms of autoimmune diseases (caused byabnormality in human immune system mistaking cells or tissues of itselffor antigens due to genetic predisposition and low molecular extrinsicfactor called hapten (for example, rheumatism (such as rheumatoidarthritis), systemic lupus erythematosus, collagenosis, multiplesclerosis, and atopies).

Also, the autoimmune agent of the present invention, has no harmful sideeffect, and thus it can be administered over a long period.

The invention provides drugs comprising the anti-inflammatory agent asan anti-inflammatory active ingredient.

The drugs of the present invention comprise periandrins, Brazilianlicorice extract, or one or more constituents contained in the extract.Thus, they have an anti-inflammatory effect.

Particularly, the drugs of the present invention reduce inflammation dueto autoimmune diseases (such as rheumatism (rheumatoid arthritis, forexample), systemic lupus erythematosus, collagenosis, multiplesclerosis, and atopies).

The anti-inflammatory agent of the present invention is alsoadvantageous in that it has no harmful side effect.

The drugs of the present invention, can be made into various forms andcompounded with other ingredients.

The invention provides cosmetics containing the anti-inflammatory agentas an anti-inflammatory active ingredient.

The cosmetics of the present invention comprise periandrins, Brazilianlicorice extract, or one or more constituents of the extract. Therefore,they have an anti-inflammatory effect (i.e. effect by which inflammationdue to autoimmune diseases such as rheumatism (rheumatoid arthritis, forexample), systemic lupus erythematosus, collagenosis, multiplesclerosis, and atopies is reduced).

Accordingly, use of the cosmetics of the present invention can reduceinflammation caused by the aforementioned symptoms.

The cosmetics of the present invention are also advantageous in thatthey have no side effect.

The cosmetics of the present invention, can be made into various formsand products.

The invention provides foods comprising the anti-inflammatory agent asan anti-inflammatory active ingredient.

The foods of the present invention comprise periandrins, Brazilianlicorice extract, or one or more constituents contained in the extract.Therefore, they have an anti-inflammatory effect (i.e. effect by whichinflammation due to autoimmune diseases such as rheumatism (rheumatoidarthritis, for example), systemic lupus erythematosus, collagenosis,multiple sclerosis, atopies, etc. is reduced).

Accordingly, an intake of the foods of the present invention, forexample, can reduce inflammation due to the aforementioned symptoms.

The foods of the present invention are also advantageous in that theyhave no harmful side effect.

The foods of the present invention, can be made into various forms andcompounded with other ingredients.

The invention provides food materials comprising the anti-inflammatoryagent as an anti-inflammatory active ingredient.

The food materials of the present invention comprise periandrins,Brazilian licorice extract, or one or more constituents contained in theextract. Therefore, they have an anti-inflammatory effect (i.e. effectby which inflammation due to autoimmune diseases such as rheumatism(rheumatoid arthritis, for example), systemic lupus erythematosus,collagenosis, multiple sclerosis, atopies, etc. is reduced).

Accordingly, an intake of the food materials of the present invention,for example, can reduce inflammation due to the aforementioned symptoms.

The food materials of the present invention can be a sweetener, forexample.

The food materials of the present invention, can be compounded withvarious ingredients and can be added to various foods.

The invention provides drugs comprising the immunosuppressant as animmunosuppressive active ingredient.

The drugs of the present invention comprise periandrins, Brazilianlicorice extract, or one or more constituents contained in the extract.Thus, they have an immunosuppressive effect.

Accordingly, the drugs of the present invention reduce development ofautoimmune diseases (such as rheumatism (rheumatoid arthritis, forexample), systemic lupus erythematosus, collagenosis, multiplesclerosis, and atopies) and also ease the symptoms thereof.

The immunosuppressant of the present invention is also advantageous inthat it has no harmful side effect.

The drugs of the present invention, can be made into various forms andcompounded with other ingredients.

The invention provides cosmetics comprising the immunosuppressant as animmunosuppressive active ingredient.

The cosmetics of the present invention comprise periandrins, Brazilianlicorice extract, or one or more constituents of the extract. Therefore,they have an immunosuppressive effect.

Accordingly, use of the cosmetics of the present invention can reducedevelopment of autoimmune diseases such as rheumatism (rheumatoidarthritis, for example), systemic lupus erythematosus, collagenosis,multiple sclerosis, and atopies, and ease the symptoms thereof.

The cosmetics of the present invention are also advantageous in thatthey have no side effect.

The cosmetics of the present invention, can be made into various formsand products.

The invention provides foods comprising the immunosuppressant as animmunosuppressive active ingredient.

The foods of the present invention comprise periandrins, Brazilianlicorice extract, or one or more constituents contained in the extract.Therefore, they have an immunosuppressive effect.

Accordingly, an intake of the foods of the present invention, forexample, can reduce development of autoimmune diseases such asrheumatism (rheumatoid arthritis, for example), systemic lupuserythematosus, collagenosis, multiple sclerosis, atopies) and ease thesymptoms thereof.

The foods of the present invention are also advantageous in that theyhave no harmful side effect.

The foods of the present invention, as well as the foods can be madeinto various forms and compounded with other ingredients.

The invention provides food materials comprising the immunosuppressantas an immunosuppressive active ingredient.

The food materials of the present invention comprise periandrins,Brazilian licorice extract, or one or more constituents contained in theextract. Therefore, they have an immunosuppressive effect.

Accordingly, an intake of the food materials of the present invention,for example, can reduce development of autoimmune diseases such asrheumatism (rheumatoid arthritis, for example), systemic lupuserythematosus, collagenosis, multiple sclerosis, atopies)

and ease the symptoms thereof.

The food materials of the present invention can be a sweetener, forexample.

The food materials of the present invention, can compound with variousingredients and can be added to various foods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an explanatory diagram of an experimentation method accordingto an experiment 1;

FIG. 2 is an explanatory diagram showing weight changes of mice in anexperiment 2;

FIG. 3 is an explanatory diagram showing changes in evaluation values ofarthritis in the experiment 2;

FIG. 4 is an explanatory diagram showing changes in foot pad swelling inthe experiment 2;

FIG. 5 is an explanatory diagram showing changes in evaluation values ofencephalomyelitis in an experiment 3;

FIG. 6 is an explanatory diagram showing weight changes of mice in anexperiment 4;

FIG. 7 is an explanatory diagram showing changes in evaluation values ofencephalomyelitis in the experiment 4; and

FIG. 8 is an explanatory diagram contrastively showing an effect ofinhibiting hydroxysteroid dehydrogenase activation as a side effect inan experiment 5.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Cytokine production inhibitors, agents for protecting and promotingliver function, anti-inflammatory agents, immunosuppressants, drugs,cosmetics, foods, and food materials according to the present inventionwill now be explained below.

a) Firstly, extract, etc. obtained in examples and comparative examplesare described.

EXAMPLE 1

Brazilian licorice extract was obtained using the following steps I-V.These steps were repeated three times.

I. 300 g of dried rootstalk of Brazilian licorice was ground, and then,extraction was performed for an hour with 900 g of 30 wt % ethanol atthe circumfluent temperature.

II. The liquid extract obtained in step I was suctioned and filtered,and then, solid-liquid separation was performed.

III. The solid material obtained in step II was again ground andextraction was performed as in step I, and then the obtained liquidextract was separated as in step II.

IV. The solid material obtained in step III was again ground andextraction was performed as in step I, and then the obtained liquidextract was separated as in step II.

V. All the liquid extracts obtained in steps I, III and IV werecollected together, vacuumed and concentrated, and then dried byspray-drying to obtain solid extract.

Average yield and standard deviation of the last extract obtained in theabove manner were, respectively, 1.03 g and 0.49 g.

This Brazilian licorice extract, as can be understood from the laterdescribed experiments, has effects of inhibiting cytokine production andprotecting and promoting liver function. Accordingly, this Brazilianlicorice extract can be used as cytokine production inhibitors, agentsfor protecting and promoting liver function, drugs, cosmetics, foods,and food materials.

EXAMPLE 2

The extraction method of Example 1 was exercised in basically the samemanner to obtain Brazilian licorice extract. However, in this case, 900g of 20 wt % ethanol was used in place of 900 g of 30 wt % ethanol forextraction.

Average yield and standard deviation of the extract obtained in theabove manner were, respectively, 1.53 g and 2.11 g.

This Brazilian licorice extract, as can be understood from the laterdescribed experiments, has an anti-inflammatory effect and animmunosuppressive effect. Accordingly, this Brazilian licorice extractcan be used as anti-inflammatory agents, immunosuppressants, drugs,cosmetics, foods, and food materials.

EXAMPLE 3

Brazilian licorice extract obtained in Example 2 was fractionated usingthe following steps I-IV.

I. 575 g of Brazilian licorice extract was dissolved in a 20 v/v %methanol solution, and introduced into a column (300×400 mm) which wasfilled with ion-exchange resin (DIAION HP-20, produced by MitsubishiChemical Corporation), to be suctioned and cleaned with a 20 v/v %methanol solution.

II. The cleaning component obtained in step I was dried under reducedpressure, and 106 g of a solid content (BL. 1) was thus obtained.

III. The column is cleaned with a 50 v/v % methanol solution, and bydrying under reduced pressure the resulted cleaning solution, 63.4 g ofa solid content (BL. 2) was obtained.

IV. The column is cleaned with 80 v/v % methanol solution, and by dryingunder reduced pressure the resulted cleaning solution, 296 g of a solidcontent (BL. 3) was obtained.

In order to prove that periandrins are contained in the BL. 3, thefollowing method was used.

Particularly, purified periandrins obtained in later describedExperiment 4 were made a preparation. Periandrins were identifiedthrough silica gel thin-layer chromatography using two types ofdeveloping solvents (one of which contains chloroform, methanol, waterand acetic acid in the proportions of 70:35:5:5; and the other of whichcontains ethyl acetate, isopropyl alcohol, water, ethanol anddiethylamine in the proportions of 20:10:8:1:0.3).

The BL. 3 (which is a constituent contained in Brazilian licoriceextract and which contains periandrins) has an excellentimmunosuppressive effect as seen in the later explained Experiment 4.

EXAMPLE 4

From the extract obtained in Example 1 or 2, purified periandrins (PD-I,PD-II, PD-III and PD-IV) were obtained by way of the usual separationpurification method using column chromatography.

Nuclear magnetic resonance spectrum and mass spectrum of the obtainedPD-I, PD-II, PD-III and PD-IV were measured respectively, and it wasfound that each of them is not a mixture.

The periandrins (PD-I, PD-II, PD-III and PD-IV) obtained in Example 4are, as shown in later explained Experiment 5, hardly inhibits activityof 11 β-hydroxysteroid dehydrogenase.

Therefore, these periandrins do not develop pseudoaldosteronism, whichis considered to be caused by inhibition of 11 β-hydroxysteroiddehydrogenase activity.

These penandrins have effects of inhibiting cytokine production andprotecting and promoting liver function, an anti-inflammatory effect,and an immunosuppressive effect.

EXAMPLE 5

From Brazilian licorice extract obtained in Example 2, purifiedperiandrins (PD-I, PD-II, PD-III and PD-IV) were obtained using thefollowing steps I-IV.

I. 4-nitrobenzylbromide (produced by Tokyo Kasei Kogyo Co., Ltd.),dimethylformaldehyde as a solvent, and triethylamine as a hydrobromicscavenger were added to Brazilian licorice extract. According to theusual manner, 4-nitrobenzylization was performed.

As a result, 4-nitrobenzylized PD-I, PD-II, PD-III and PD-IV wereobtained.

II. After the 4-nitrobenzylization in step I, dimethylformaldehyde wasvacuumed and distilled, to extract a reaction product containing4-nitrobenzylized PD-I, PD-II, PD-III and PD-IV with ethyl acetate.

III. From the ethyl acetate extract obtained in step II, the4-nitrobenzylized PD-I, PD-II, PD-III and PD-IV were separated andpurified through chromatography with silica gel (WAKOGEL C-200, producedby Wako Pure Chemical Industries,Ltd.) and octadecylsilane (produced byChromatorex, Fuji Silysia Chemical Ltd.).

IV. The 4-nitrobenzylized PD-I, PD-II, PD-III and PD-IV obtained in stepII were dissolved in an acetic solvent, and then, by adding zinc powder,denitrobenzilized reaction was performed to obtain purified periandrins(PD-I, PD-II, PD-III and PD-IV).

The periandrins obtained in Example 5 are, like the periandrins obtainedin Example 4, hardly inhibits activity of 11 β-hydroxysteroiddehydrogenase and thus do not develop pseudoaldosteronism.

Furthermore, the periandrins obtained in Example 4 or 5 have, as can beclearly seen from later explained Experiment 6, effects of inhibitingcytokine production and protecting and promoting liver function, ananti-inflammatory effect and an immunosuppressive effect.

Comparative Example 1

Chinese licorice extract was obtained in the same manner as in theaforementioned Example 1.

Chinese licorice is a typical species of licorice, which has been usedas an agent for protecting liver function.

Average yield and standard deviation of the extract obtained in theabove manner were, respectively, 27.0 g and 1.03 g.

Comparative Example 2

Chinese licorice extract was obtained in the same manner as theaforementioned Example 2.

Average yield and standard deviation of the extract obtained in theabove manner were, respectively, 28.5 g and 2.18 g.

b) Secondly, experiments conducted to confirm the effects of the extractobtained in the aforementioned Examples are explained.

Experiment 1

The following experiment was performed to examine the effects ofinhibiting cytokine production and lowering GOT activity of the extractsobtained in Example 1 and Comparative Example 1. The experimentprocedures are shown in FIG. 1.

I. Preparation of Mice to be Used in the Experiment

Male mice of ddy system (Japan SLC, Inc.), each of which weighs 18-20 g,are prepared for the experiment. The mice are divided into six groups,namely, A, B, C, D, E and F, each consisting of eight mice.

II. Injection of Propionivacterium Acnes Suspension

5 mg of Propionivacterium acnes suspension was injected into the vein ofthe aforementioned mice respectively, to cause hepatopathy.

III. Injection of Brazilian Licorice Extract and Comparative Sample

A solvent without drugs was injected into the mice in group A.

Brazilian licorice extract obtained in Example 1 was injected into theabdominal cavity of the mice in groups B and C four times in total, thatis, one day, three days, five days and seven days after the injection ofthe propionivacterium acnes suspension in step II. The dosage per oneinjection was 25 mg, respectively.

Chinese licorice extract obtained in Comparative Example 1 was injectedinto the abdominal cavity of the mice in groups D and E four times intotal, that is, one day, three days, five days and seven days after theinjection of the propionivacterium acnes suspension in step II. Thedosage per one injection was 25 mg, respectively.

5 mg of prednisolone acetate was injected into the abdominal cavity ofthe respective mice in group F four times in total, that is, one day,three days, five days and seven days after the injection of thepropionivacterium acnes suspension in step II.

Prednisolone acetate is a typical type of prednisolone, which has beenused as a cytokine production inhibitor.

IV. Sampling of Blood

10 μg of lipopolysaccharide was injected into the vein of the mice inall groups respectively, thirty minutes after the fourth injection instep II (as to the mice in group A, the injection was performed at thesame timing as the mice in the other groups). Then, two hours later,blood was taken from all the mice to obtain serum. There was no visiblechange in weight and food and water consumption of the mice.

V. Measurement of Cytokine in Blood

From the serum obtained in step III, measurement of interleukin-6(IL-6), which is a kind of cytokine, was performed.

For the measurement, a commercially available measurement kit whichadopts Fluorescence Linked immuno-Sorbent Assay using fluorescencelabeling antibody was used.

VI. Measurement of GOT in Blood

From the serum obtained in step III, glutamic-oxaloacetic transaminaseactivity (GOT) was measured.

For the measurement, a commercially available measurement kit whichadopts Enzyme Linked immuno-Sorbent Assay using enzyme labeling antibodywas used.

Blood cytokine (IL-6) levels of the respective eight mice were measuredper each of the groups A-F. Table 1 shows the average yield and standarddeviation.

The measured values in Table 1 show IL-6 levels (unit: picogram) per 1ml serum.

TABLE 1 Average of Standard Group eight mice deviation A 360349 93784 B40845 11348 C 76772 58125 D 280986 97265 E 191646 91081 F 26473 15300

As shown in Table 1, the blood IL-6 levels of the mice in groups B and Cto which Brazilian licorice extract was injected were remarkably reducedcompared to the blood IL-6 levels of the mice in group A without druginjection and of the mice in groups D and E to which Chinese licoriceextract was injected. The levels were rather close to the blood IL-6levels of group F to which prednisolone acetate was injected.

In view of the above, it is clear that Brazilian licorice extract has aneffect of inhibiting cytolkine production which equals to the effect byprednisolone acetate.

Table 2 shows average yield and standard deviation of blood GOT activitylevels of the respective eight mice measured per each of the groups A-F.

The measured values in Table 2 are shown in Kermen Units.

TABLE 2 Average of Standard Group eight mice deviation A 918.160 256.863B 244.359 31.405 C 201.840 18.893 D 462.386 118.867 E 412.544 159.981 F350.705 68.166

As can be seen in Table 2, the blood GOT activity levels of the mice ingroups B and C to which Brazilian licorice extract was injected werelower than the blood GOT activity levels of any other groups.

In view of the above, Brazilian licorice extract has a superior effectof protecting and promoting liver function to Chinese licorice extractor prednisolone acetate.

Experiment 2

Anti-inflammatory effect and immunosuppressive effect of the extractobtained in Example 2 and Comparative Example 2 were examined by meansof collagen induction arthritis model experiment.

I. Preparation of Mice to be Used in Experiment

DBJ/1J mice were used in the experiment. The mice are divided into sixgroups, namely, G, H, I, J, K and L, each consisting of six mice.

II. Induction of Arthritis by Injection of Emulsion to Mice (Immunity)

An equal amount of 8 mg/ml concentration of a collagen solution (thesolvent is phosphate buffered saline containing 0.01 mol/L acetic acid)and fetal bovine serum containing 4 mg/ml concentration ofsupersonically treated Microbacterium tuberculosis H37Ra were mixed toprepare emulsion.

50 μl (200 μg/head) of the emulsion was then injected to the back neckskin of each of the mice under ether (first immunity).

Three weeks after the first immunity, 50 μl (200 μg/head) of theemulsion was injected to the tail head skin of each of the mice (secondimmunity).

Arthritis was induced in the respective mice due to the first and secondimmunities.

III. Injection of Brazilian Licorice Extract and Comparative Sample toMice

A solvent without drugs was injected into the mice in group G.

Brazilian licorice extract obtained in Example 2 was diluted byphysiological saline so that the dosage is 6 mg/head, and the obtaineddiluted solution was injected into the abdominal cavity of the mice ingroup H by 1 ml every other day after the first immunity.

Brazilian licorice extract obtained in Example 2 was diluted byphysiological saline so that the dosage is 12.5 mg/head, and theobtained diluted solution was injected into the abdominal cavity of themice in group I by 1 ml every other day after the first immunity.

Chinese licorice extract obtained in Comparative Example 2 was dilutedby physiological saline so that the dosage is 6 mg/head, and theobtained diluted solution was injected into the abdominal cavity of themice in group J by 1 mg every other day after the first immunity.

Chinese licorice extract obtained in Comparative Example 2 was dilutedby physiological saline so that the dosage is 12.5 mg/head, and theobtained diluted solution was injected into the abdominal cavity of themice in group K by 1 mg every other day after the first immunity.

Prednisolone acetate (produced by Shionogi & Co., Ltd.) which is acontrol drug was suspended by physiological saline so that the dosage is5 mg/Kg of body weight, and the obtained suspended solution was injectedinto the abdominal cavity of each mouse in group L by 1 ml every otherday after the first immunity.

The above injections were performed all through the test period (foreight weeks after the first immunity).

IV. Weight Measurement of Mice

The weight of the mice in the respective groups were measured every dayafter the first immunity and the average weight per each of the groupswas calculated. The results are shown in FIG. 2.

As can be seen from FIG. 2, there was no visible reduction of averageweight in the mice of groups H and I to which Brazilian licorice extractwas injected. In addition, there was no reduction in average weight inthe mice of groups H and I after the second immunity, either.

On the other hand, the average weights of the mice in group G withoutdrug injection, groups J and K to which Chinese licorice extract wasinjected, and group K to which prednisolone acetate was injected werereduced significantly compared to the average weights of mice in groupsH and I.

V. Accordingly, Brazilian licorice extract hardly causes side effectsand is highly secure compared to Chinese licorice extract andprednisolone acetate.

Evaluation of Arthritis Condition (Arthritis Index)

Clinical symptoms of arthritis of mice in the respective groups wereevaluated by one and the same observer using 6 rankings from zero tofive (the larger the value is, the worse the symptom is), and theaverages per the respective groups were calculated. These evaluationswere performed in 0, 3^(rd), 4^(th), 5^(th), 6^(th), 7^(th) and 8^(th)week after the first immunity The results were shown in FIG. 3 and Table3. In Table 3, values in the upper section show the average in therespective groups, and the values in the lower section show the standarddeviation.

TABLE 3 Primary Drug type Gr. Dosage immunity 3rd week 4th week 5th week6th week 7th week 8th week None G — 0 0.688 1.313 3.125 2.688 2.6882.688 0 0.377 0.4 0.611 0.582 0.49 0.462 B.L. H   6 mg/ 0 0 0.25 1.6251.625 0.938 0.929 head 0 0 0.189 0.653 0.673 0.29 0.254 I 12.5 mg/ 0 0 00.9 1.2 0.8 0.6 head 0 0 0 0.9 0.846 0.339 0.367 C.L. J   6 mg/ 0 0 0.52.833 2.75 2.583 2.167 head 0 0 0.5 0.843 0.588 0.396 0.307 K 12.5 mg/ 00 0.571 3 3.071 3.071 2.357 head 0 0 0.277 0.556 0.539 0.297 0.404 Pred.L   5 mg/ 0 0 0 0 0 0.125 0.125 head 0 0 0 0 0 0.125 0.125

As can be seen from FIG. 3 and Table 3, the arthritis symptoms of micein groups to which Brazilian licorice extract was injected (groups H andI) were improved compared to the mice in group G without drug injectionand groups J and K to which Chinese licorice extract was injected.

In view of the above, it is clear that Brazilian licorice extract hassuperior anti-inflammatory and immunosuppressive effects to Chineselicorice extract.

As to the mice in groups H and I to which Brazilian licorice extract wasinjected, the symptoms of the mice in group I to which large quantity(12.5 mg/head) of the extract was injected per one time were betterimproved than the mice in group H to which small quantity (6 mg/head) ofthe extract was injected per one time. In short, the effect of improvingarthritis symptoms by Brazilian licorice extract showed dose dependency.

VI. Measurement of Foot Pad Swelling (Foot Pad Volume)

The left and right hind foot pad volumes of the mice in the respectivegroups were measured using a plethysmometer (TK-101 UNICOM), and theaverage per each of the groups was calculated. These evaluations wereperformed in 0, 3^(rd), 4^(th), 5^(th), 6^(th), 7^(th) and 8^(th) weeksafter the first immunity. The results were shown in FIG. 4 and Table 4.The values in FIG. 4 are relative values based on the initial hind footpad volumes. In Table 4, the values in the upper section show theaverages of the respective groups and the values in the lower sectionshow the standard deviation.

TABLE 4 Primary Drug type Gr. Dosage immunity 3rd week 4th week 5th week6th week 7th week 8th week None M — 100 112.9 111 143.3 141.2 124.7122.7 0 4.517 6.084 6.821 5.534 3.109 2.241 B.L. N   6 mg/ 100 107.9 104123.1 126.4 114 114.7 head 0 1.34 1.871 6.793 5.543 3.117 2.509 O 12.5mg/ 100 107.3 101 117.8 121.7 116 112.4 head 0 3.588 2.527 6.113 6.043.417 2.478 C.L. P   6 mg/ 100 100.9 100.7 130 148.6 124.6 123.4 head 03.644 4.638 12.752 8.55 4.338 4.846 Q 12.5 mg/ 100 105.4 101.5 150.8130.7 122.2 124.4 head 0 1.287 3.145 7.663 6.619 4.528 4.313 Pred. R   5mg/ 100 99.2 95.2 101.1 102.4 95.5 97.9 head 0 2.328 2.037 2.347 1.8661.615 1.692

It is clear from FIG. 4 and Table 4 that there was a superior effect ofinhibiting foot pad swelling in the mice in groups H and I to whichBrazilian licorice extract was injected compared to the mice in group Gwithout drug injection and groups J and K to which Chinese licoriceextract was injected.

It should be noted that a superior effect of inhibiting foot padswelling was seen even in the mice in group I to which less Brazilianlicorice extract (6 mg/head) was injected compared to the mice in groupsG, J and K.

In view of the above, Brazilian licorice extract has superioranti-inflammatory and immunosuppressive effects to Chinese licoriceextract.

Experiment 3

Anti-inflammatory effect and immunosuppressive effect of the extractobtained in Example 2 and Comparative Example 2 were examined byautoimmune encephalomyelitis model experiment using rats.

I. Preparation of Rats to be Used in Experiment

Eight weeks old male rats of DA system were used in the experiment. Therats were divided into 6 groups, namely, M, N, O, P, Q and R, eachconsisting of six rats.

II. Induction of Autoimmune Encephalomyelitis by Injection of Emulsionto Rats

An equal amount of 2 mg/ml concentration of a bovine Myeline BasicProtein (MBP) solution (the solvent is phosphate buffered saline) andfetal bovine serum containing supersonically treated 4 mg/mlMicrobacterium tuberculosis H37Ra were mixed to prepare emulsion.

50 μl of the emulsion was then injected hypodermically to hind leg footpads of the respective rats under ether (in other words, 100 μl (100μgMBP/head) was injected per rat), to induce autoimmuneencephalomyelitis.

III. Injection of Brazilian Licorice Extract and Comparative Sample

A solvent without drugs was injected into the rats in group M.

Brazilian licorice extract obtained in Example 2 was diluted byphysiological saline so that the dosage is 25 mg/head, and the obtaineddiluted solution was injected into the abdominal cavity of the rats ingroup N by 1 ml once a day for 12 consecutive days after the injectionof emulsion.

Brazilian licorice extract obtained in Example 2 was diluted byphysiological saline so that the dosage is 50 mg/head, and the obtaineddiluted solution was injected into the abdominal cavity of the rats ingroup O by 1 ml once a day for 12 consecutive days after the injectionof emulsion.

Chinese licorice extract obtained in Comparative Example 2 was dilutedby physiological saline so that the dosage is 25 mg/head, and theobtained diluted solution was injected into the abdominal cavity of therats in, group P by 1 ml once a day for 12 consecutive days after theinjection of emulsion.

Chinese licorice extract obtained in Comparative Example 2 was dilutedby physiological saline so that the dosage is 50 mg/head, and theobtained diluted solution was injected into the abdominal cavity of therats in group Q by 1 ml once a day for 12 consecutive days after theinjection of emulsion.

Prednisolone acetate (produced by Shionogi & Co., Ltd.) which is acontrol drug was suspended by physiological saline so that the dosage is5 mg/Kg of body weight, and the obtained suspended solution was injectedinto the abdominal cavity of each rat in group R by 1 ml once a day for12 consecutive days after the injection of emulsion.

IV. Evaluation of Encephalomyelitis (Clinical Score)

After the injection of emulsion, the rats in the respective groups wereobserved every day to evaluate symptoms of encephalomyelitis (Clinicalsore). The symptoms were evaluated by six rankings from zero to five(the larger the value, the worse the symptom is) according to thecriteria shown in Table 5, and the averages per the respective groupswere calculated. The above observations were conducted for 25consecutive days after the injection of emulsion.

TABLE 5 Value Criteria of clinical score of encephalomyelitis 0 Nochange 1 Paralysis of tail 2 Incomplete paralysis of hind legs 3Complete paralysis of hind legs and incomplete paralysis of front legs 4Paralysis of limbs, incontinence 5 Dead

The results of the evaluation are shown in FIG. 5. As can be clearlyseen from FIG. 5, a few rats in group N to which 25 mg/head of Brazilianlicorice extract was injected developed encephalomyelitis during andafter the injections. However, most of the rats did not developencephalomyelitis.

The mice in group O to which 50 mg/head of Brazilian licorice extractwas injected did not develop encephalomyelitis during the injections atall. However, they developed encephalomyelitis after the injections werecompleted.

On the other hand, the rats in group M without drug injection was andgroups P and Q to which Chinese licorice extract was injected developedencephalomyelitis 7-8 days after the injection of emulsion. The symptomsreached its peak on 11^(th)-12^(th) days and were disappeared on 14^(th)day. The symptoms of the rats in group Q to which more Chinese licoriceextract was injected (50 mg/head) were slightly better than the rats ingroups M and P.

A few rats in group R to which prednisolone acetate was injecteddeveloped encephalomyelitis 8-11 days after the injection of emulsion.

In view of the above, Brazilian licorice extract has superioranti-inflammatory and immunosuppressive effects to Chinese licoriceextract.

Experiment 4

The immunosuppressive effect of the BL. 1, BL. 2 and BL. 3, which arefractionated components of Brazilian licorice extract obtained inExample 3, was examined by autoimmune encephalomyelitis model experimentusing rats.

The manner of experiment was basically the same as the aforementionedExperiment 3.

However, the number of groups of rats were reduced to five, one of whichincluded the rats to which the BL. 1 was injected, another of whichincluded the rats to which the BL. 2 was injected, another of whichincluded the rats to which the BL. 3 was injected, another of whichincluded the rats to which prednisolone acetate was injected, andanother of which included the rats without injection.

The dose per one injection of BL. 1, BL. 2 and BL. 3 was 6 mg/head andin the form of physiological saline.

The dose per one injection of prednisolone acetate was 5 mg/Kg of bodyweight and in the form of physiological saline.

FIG. 6 shows weight changes of the rats in the respective groups duringthe experiment period.

The rats in the groups to which the BL. 1, BL. 2 and BL. 3 were injectedgained about additional 5-10 g compared to the rats in the control groupon and after 8^(th) day after the immunity.

The rats in the group to which prednisolone acetate was injected had theleast increase among the rats in the other groups.

FIG. 7 shows the evaluation results on encephalomyelitis. The evaluationcriteria are the same as the above Table 5.

Development of encephalomyelitis was strongly inhibited in the rats towhich the BL. 3 was injected compared to any of the rats in the groupsto which the BL. 1 and BL. 2 were injected, and the rats in the controlgroup. Development of encephalomyelitis was slightly inhibited in therats to which the BL. 2 was injected compared to the rats in the controlgroup.

In addition, there was no temporary development (for example,development caused on 15^(th)-16^(th) days in the rats of group O inExperiment 3) after completion of the injections in the rats in thegroup to which the BL. 3 was injected.

The results of Experiment 4 indicate that not the component contained inthe BL. 1 (polar component such as polysaccharide which is not held bysuction chromatography carrier such as HP-20), but the polar componentsuch as triterpenoid glycoside (for example, periandrins) contained inthe BL. 3 has an immunosuppressive effect.

Experiment 5

In order to evaluate the effect of inhibiting 11 β-hydeoxysteroiddehydrogenase activity by the purified periandrins (PD-I, PD-II, PD-III,PD-IV) obtained in Example 4 or 5, IC 50 values (concentration in whichthe 50 percent of activity of the above enzyme is inhibited) of thepurified periandrins were measured by the following steps I-VI.

Note that inhibition of 11 β-hydroxysteroid dehydrogenase activity isconsidered to be a cause of pseudoaldosteronism.

I. An enzymatic solution containing rat liver microsome as a preparationof 11 β-hydroxysteroid dehydrogenase was prepared as below.

Fresh rat liver was ice-cooled and homogenized in 0.25 M sucrosesolution. Then, at the temperature of 4° C., it was centrifuged for 20minutes at 10,000×g.

After separating cell organelles of the nucleus, mitochondria andlysosome as deposits, the supernatant layer was further centrifuged at105,000×g for 60 minutes at the temperature of 4° C. to obtain amicrosome fraction as a precipitate.

The obtained microsome fraction was suspended by 0.25 M sucrose solutionto produce an enzymatic solution, and the obtained solution was storedat the temperature of −20 degrees till the time of measurement of enzymeactivity.

II. Measurement of Enzyme Activity

1 mM of NADP (product of Oriental Yeast Co., Ltd,) as a coenzyme and anamount of PD-I having a predetermined concentration (ranging from 2 to1480 μM) as an inhibitor were added to 0.8 ml of Tris-HCI buffersolution having the concentration of 100 mM (PH8.0).

0.2 ml of the enzymatic solution prepared in step I was added to theobtained solution, and after three minutes of preincubation at 37° C.,0.02 ml of 5 mM hydrocortisone solution which was dissolved in methanolwas added to start a reaction between 11 β-hydroxysteroid dehydrogenaseand PD-I. The reaction was continued for 30 minutes at the temperatureof 30° C., and then it was stopped by adding 0.5 ml of ethyl acetate.

This solution was centrifuged and an ethyl acetate layer was separatedinto an Eppendorf tube. In the mean time, cortisone and substratehydrocortisone generated by the reaction between 11 β-hydroxysteroiddehydrogenase and substrate hydrocortisone are transferred to the ethylacetate layer.

Furthermore, the water phase after the centrifuge was again centrifugedin the same manner as above by adding 0.5 ml of ethyl acetate toseparate an ethyl acetate layer. This process was repeated two moretimes.

The ethyl acetate layers obtained by three time extraction were puttogether into an Eppendorf tube and dried using a centrifugalevaporator.

III. Determination of Generated Cortisone

To prepare a measurement sample, 0.2 ml of methanol was added into theEppendorf tube in which the ethyl acetate layers were dried in step II,and then the tube was left for a night so that cortisone was completelydissolved. Cortisone was determined using HPLC.

Particularly, quantity of cortisone contained in the sample wascalculated from a ratio of a peak area corresponding to cortisone whenthe measurement sample was introduced to HPLC to a peak area when 5 μlof standard solution (0.1 mg/ml of cortisone-methanol solution) wasintroduced.

The measurement conditions of HPLC were as follows.

Stationary phase: NovaPak cartridge C18, 3.9 × 150 mm (product of JapanWaters) Moving phase: 45% methanol solution containing 0.1%trifluoroacetic acid (isocratic) Flow rate: 0.5 ml/min Detection: UV 245nm Injection amount: 5 μl

IV. Relative activity (ratio of activity remained after reaction with aninhibitor to the initial activity) of 11 β-hydroxysteroid dehydrogenasewas calculated from the quantity of cortisone in accordance with thefollowing equation.Relative activity=((C _(I) −C _(BL))/(C _(T) −C _(BL)))×100(%)

-   -   where    -   C_(T): cortisone quantity generated when no inhibitor is added;    -   C_(I): cortisone quantity generated when an inhibitor exists;        and    -   C_(BL): cortisone quantity when enzyme is not added.

V. Measurement of relative activity when PD-II, PD-III, PD-IV, andglycirrhizin were added as an inhibitor instead of PD-I was performed inthe same manner as in steps I-IV.

The concentration of the inhibitor PD-I, PD-II, PD-III or PD-IV in themeasurement of relative activity is a predetermined value ranging from 2to 1480 μM. The concentration of glycirrhizin is a predetermined valueranging from 0.2 to 445 μM.

The system to which no inhibitor was added was considered as a control,and the system to which no substrate was added is considered as a blank.

VI. As shown in FIG. 6, relation between the concentrations of therespective inhibitors and the relative activity was plotted, and theinhibitor concentration of which relative activity is equal to 50% wasmade IC50 of the inhibitor.

As to the measurement results, on one hand, the IC50 value ofglycirrhizin was 26.2 μM, and on the other hand, the IC50 values ofPD-I, PD-II, PD-III and PD-IV were respectively 214 μM, 150 μM, 581 μM,240 μM, which were high in one order.

In other words, inhibitory activity of PD-I, PD-II, PD-III and PD-IV wasabout one fifth to one twentieth of glycirrhizin.

Accordingly, since the purified periandrins hardly inhibit 11β-hydroxysteroid dehydrogenase activity, pseudoaldosteronism, which isconsidered to be caused by inhibition of 11 β-hydroxysteroiddehydrogenase activity, is not developed by using the productscomprising the purified periandrins (cytokine production inhibitors,agents for protecting and promoting liver function, anti-inflammatoryagents, immunosuppressants, drugs, cosmetics, foods and food materials).

Experiment 6

The effects of inhibiting cytokine production and of protecting andpromoting liver function with respect to PD-I, PD-II, PD-III and PD-IVobtained in Example 4 or 5 were examined in the same manner as inExperiment 1.

In this case, however, the number of groups of rats were made six, oneof which includes the rats to which PD-I was injected, another of whichincludes the rats to which PD-II was injected, another of which includesthe rats to which PD-III was injected, another of which includes therats to which PD-IV was injected, another of which includes the rats towhich prednisolone acetate was injected, and another of which includesthe rats to which a solvent without any drugs was injected.

The dose per one injection of PD-I, PD-II, PD-III and PD-IV was 0.1-3mg/Kg of body weight.

The dose per one injection of prednisolone acetate was 5 mg/Kg of bodyweight.

All of PD-I, PD-II, PD-III and PD-IV show the effects of inhibitingcytokine production and protecting and promoting liver function. It wasfound that these components are at least one part of the activeingredient of Brazilian licorice extract.

Furthermore, the collagen induction arthritis model experiment andautoimmune encephalomyelitis model experiment shown in Experiments 2 and3 were applied using PD-I, PD-II, PD-III and PD-IV obtained inExperiment 4. As a result, these components eased the symptoms ofarthritis and encephalomyelitis like Brazilian licorice extract did.

From the above, the purified periandrins (PD-I, PD-II, PD-III and PD-IV)have anti-inflammatory effect and immunosuppressive effect.

The present invention should not be limited to the describedembodiments, and other modifications and variations might be possiblewithout departing from the scope of the invention.

For example, in the process of obtaining Brazilian licorice extract, thesolvent is not limited to ethanol, but other solvents (e.g. water,alcohols such as methanol, isopropanol, isobutanol, hexanol, methyl amylalcohol, 2-ethyl butanol, n-octyl alcohol, etc., polyhydric alcohols andthe derivatives such as glycerin, ethylene glycol, ethylene glycolmonomethyl ether, ethylene glycol monoethyl ether, propylene glycol,propylene glycol monomethyl ether, propylene glycol monoethyl ether,triethylene glycol, 1,3-butylene glycol, hexylene glycol, etc., ketonessuch as acetone, methyl ethyl ketone, methyl isobutyl ketone, etc.,esters such as ethyl acetate, isopropyl acetate, etc., ethers such asethylether, isopropylether, n-bytylether, etc., aliphatic hydrocarbonssuch as petroleum ether, n-hexane, n-pentane, n-butane, n-octane,cyclohexane, etc., and non polar solvents such as tetrachloromethane,chloroform, trichloroethylene, benzol, toluene, etc.) can be used.

INDUSTRIAL APPLICABILITY

By adopting Brazilian licorice extract or periandrins as a cytokineproduction inhibitor, it is possible to inhibit inflammation of variousdiseases such as rheumatoid arthritis. Brazilian licorice extract andperiandrins can be used as an agent for protecting and promoting liverfunction, an anti-inflammatory agent, and an immunosuppressant.Furthermore, foods, cosmetics, sweeteners and food materials comprisingBrazilian licorice extract also have the same effect as above.

1. A method of treating an inflammatory condition in a mammalian patientin need of such treatment, comprising administering to the patient oneor more periandrins in an amount effective as an anti-inflammatory. 2.The method of treating an inflammatory condition as set forth in claim1, wherein the one or more periandrins are administered as apharmaceutical composition comprising purified Brazilian licoriceextract containing periandrins in a pharmaceutically acceptable carrieror diluent.
 3. The method of treating an inflammatory condition as setforth in claim 2, wherein the one or more periandrins are insubstantially pure form.